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Activity-Based Profiling of Proteases

Laura E. Sanman1 and Matthew Bogyo1,2,3

Departments of 1Chemical and Systems Biology, 2Microbiology and Immunology, and 3Pathology, Stanford University School of Medicine, Stanford, California 94305-5324; email: [email protected]

Annu. Rev. Biochem. 2014. 83:249–73 Keywords The Annual Review of Biochemistry is online at biochem.annualreviews.org activity-based probes, proteomics, mass spectrometry, affinity handle, fluorescent imaging This article’s doi: 10.1146/annurev-biochem-060713-035352 Abstract Copyright c 2014 by Annual Reviews. All rights reserved Proteolytic enzymes are key signaling molecules in both normal physi- Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the ap- plication of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.

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gens that contain inhibitory prodomains that Contents must be removed for the protease to become active. Proteases that are not synthesized with INTRODUCTION...... 250 inhibitory prodomains often require cofactor STRATEGIES FOR PROFILING binding or posttranslational modification for PROTEASEACTIVITY...... 250 activation. After activation, endogenous protein NaturalSubstrateTurnover...... 251 inhibitors, changes in subcellular localization, Substrate-Based Probes ...... 253 and degradation can limit protease activity Activity-BasedProbes...... 254 (Figure 1). Given the high level of posttransla- DESIGNING ACTIVITY-BASED tional regulation, it is not surprising that mea- PROBESFORPROTEASES...... 255 sures of protease activity, rather than traditional Reactive Functional Groups ...... 255 transcriptomic or proteomic analyses, are often Recognition Sequence Design ...... 258 necessary to understand biological function ReporterTags...... 259 (4). The purpose of this review is to highlight APPLICATIONS OF recent methods that allow dynamic measure- ACTIVITY-BASED PROFILING ment of protease activity in complex biological TOPROTEASES...... 261 systems. We emphasize the relatively new, but Protease Enzymology ...... 261 rapidly growing, field of activity-based pro- Drug Discovery and Development . . 263 filing, which has proven valuable for detailed Profiling Proteases in Disease ...... 265 analyses of proteases and other enzyme classes.

INTRODUCTION STRATEGIES FOR PROFILING The basic definition of a protease is an enzyme PROTEASE ACTIVITY that hydrolyzes peptide bonds in proteins. Ap- The most direct method of tracking protease proximately 700 enzymes, or roughly 2% of the activity is monitoring hydrolysis of a substrate. human genome, have known or putative prote- Turnover of both natural protein substrates olytic activity (1). Although all share the ability and substrate-based probes can provide a direct to catabolize proteins, these enzymes vary in measure of protease activation events (5). The almost every other parameter, including size, quantity of peptide fragments that are pro- localization, and quaternary structure. For sim- duced by substrate proteolysis reflects levels of plification, proteases are grouped into families protease activity, but these substrate fragments on the basis of catalytic mechanism. In humans, must be isolated from their native environ- there are five protease families: aspartyl, cys- ment for identification and quantification. In

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org teine, metallo-, serine, and threonine proteases. contrast, substrate-based probes are reagents Cysteine, serine, and threonine proteases use that are designed to directly generate a signal

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. nucleophilic active-site residues to hydrolyze upon cleavage (6). Protease activity can also peptide bonds, whereas aspartyl and metallo- be indirectly monitored using activity-based proteases use active-site residues to activate wa- probes (ABPs), which contain a substrate-like ter molecules for nucleophilic attack (2). region but, rather than being processed into Proteolytic activity is central to many fragments, covalently bind to the active pro- necessary biological processes, including tease in an enzyme-catalyzed reaction (6–8). development, differentiation, and the immune Thus, the extent of target protease modifica- response (3). However, due to the irreversible tion reports levels of activity. Each of these nature of proteolysis, proteolytic activity must strategies for determining protease activity has be highly regulated within cellular systems. been applied in diverse biological systems, and Most proteases are translated as inactive zymo- each has its own strengths and weaknesses.

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abc

Protease inhibitor PTMs Inhibition

Inactive zymogen Active protease

Environment

Proteolytic prodomain removal Degradation

Figure 1 Proteases are often synthesized as inactive zymogens (left) that are activated by several mechanisms, including posttranslational modifications (PTMs), cofactor binding, pH change, and proteolytic prodomain removal (center). Prodomain hydrolysis ( yellow stars) can occur at a macromolecular signaling complex (top), by the action of another individual protease (middle), or by autocatalysis (bottom). After activation, protease activity can be limited by endogenous inhibitors or by degradation by the proteasome (right).

Below, we discuss examples of applications and Through the use of this method, many proteins the relative merits of each strategy. can be simultaneously monitored. Gel-free enrichment methods for proteolytic peptides have also been improved, allowing for simul- Natural Substrate Turnover taneous identification and quantification of Active proteases cleave protein substrates. The the majority of substrate hydrolysis events in a most direct example of this process is autopro- given sample (5, 12). These enrichments capi- teolysis, during which a protease catalyzes its talize on the unique α-amino group exposed by own processing to become active (9, 10). The substrate proteolysis. Proteolytic peptides can simplest measure of turnover of individual sub- be enriched for by negative selection, as in the strates of interest uses sodium dodecyl sulfate COFRADIC (13) and TAILS (14) methods, or polyacrylamide gel electrophoresis (SDS- by positive selection, as with selective α-amine PAGE) to resolve proteins and fragments, fol- biotinylation by subtiligase (15) or selective lowed by Coomassie staining or immunoblot- lysine capping using O-methylisourea followed

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ting for protein visualization. However, this by biotinylation of α-amines (16). procedure requires a priori knowledge of Increasing coverage of the proteolytic

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. the substrates to be measured. Alternatively, “peptidome” by use of gel-based and gel-free turnover of all protease substrates can be mea- enrichment methods has facilitated systems- sured simultaneously by mass spectrometry level monitoring of protease activity. The (Figure 2). For example, in a technique named biological pathways into which protein sub- PROTOMAP, a proteome sample is prepared strates fall suggest protease function, and before and after addition of a protease enzyme. common cleavage sequences indicate substrate The samples are then resolved by SDS-PAGE, recognition motifs. Both have been useful for and the locations of all proteins and protein separating the biological functions of closely fragments are mapped in the gel by mass spec- related proteases and advancing the design trometry. Changes in the migration of protein of selective substrate-based probes and ABPs. fragments indicate substrate proteolysis (11). The following examples illustrate how methods

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abc

R O R1 Substrate 3 H N OH SH C terminus N N H O H O R2 R O R1 O R Substrate 3 H H –2 H Substrate + C terminus N N N N N N N terminus O R–2 H OOR H R H O H 2 –1 H N Substrate 2 N N N terminus Full-length substrate R H O Intensity –1 m/z Cleavage products Readouts

Figure 2 Cleavage of substrate proteins reports protease activation. (Left) A cysteine protease (gray; active-site sulfhydryl group represented by –SH) hydrolyzes an amide bond, releasing two polypeptides (middle). (Right) Potential measures of substrate truncation include (top right) immunoblotting or Coomassie stain as well as (bottom right) mass spectrometry. Abbreviation: m/z, mass-to-charge ratio.

of assessing natural substrate turnover have recombinant caspases-3, -7, -8, and -9 also

been used to characterize global proteolysis by generated catalytic efficiency values (kcat/KD) a single family of proteases, the caspases. for ∼500 protein substrates. Small but impor- Apoptosis, an immunologically silent form tant differences in substrate specificity among of programmed cell death, is an event initiated the caspases were determined (21) and used to and executed by the caspase family of cysteine identify substrates specific for caspases-3, -7, proteases (17). Because apoptosis is tightly and -8. These studies demonstrate the value regulated by proteolysis, many methods of of systems-level analysis of proteolysis for pro- dynamically monitoring protease activity, viding information about the types of pathways including measuring hydrolysis of natural sub- that may be regulated by proteases and about strates, have been used to study this process. protease substrate specificity. However, note Cleavage of Bid, an important regulatory pro- that although such global methods produce tein that is cleaved by caspases during apoptosis, many valuable leads, it is often difficult to is a common measure of caspase activation (18). validate an extensive list of substrates. Although Bid is a useful reporter of cell death, A major advantage of measuring protease it can be cleaved by multiple proteases (19). In activation by monitoring natural substrate addition, Bid cleavage occurs in only one of the turnover is that the methods are highly adapt- many pathways that are regulated by caspases able. In fact, mass spectrometry–based identifi-

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org during apoptosis (17–19). To profile caspase cation of proteolytic peptides has been used as activation comprehensively, a series of recent a measure of protease activity in the clinic (24–

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. studies used subtiligase-mediated proteolytic 26). For example, a recent proof-of-concept peptide enrichment and PROTOMAP to comparison of sera from prostate, bladder, and identify and quantify ∼1,300 putative caspase breast cancer patients showed that proteolytic substrates in cells and cell lysates treated with peptide signatures can predict cancer type (24). recombinant caspases or caspase-activating A disadvantage of using substrate cleavage as agents (11, 20–23). These studies established a proxy for protease activity is that it relies that, during apoptosis, caspases cleave predom- on the assumption that only a single protease inantly proteins involved in transcriptional is capable of cleaving each substrate protein. regulation, chromosomal structure, and cy- This assumption is often not true, especially toskeletal maintenance (11, 20). Quantifying in large, closely related protease families such hydrolysis over time in cell lysates treated with as the matrix metalloproteases (MMPs) (27),

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a Signal quenched b Signal released c Detection methods

O R Intensity R3 H 1 SH N OH Time N N H H R O R O R OOR2 3 H 1 H –2 H N N N N N N + H H H OOR2 R–1 O O R –2 H H N 2 N N Cleavage sequence H R–1 O

Figure 3 A sample substrate-based probe in which a fluorophore (lightbulb) and a quencher (red octagon) flank the substrate region. When intact, the probe is not fluorescent (left). When the protease is activated, the substrate is cleaved and fluorescence is released (middle). Detection of signal from substrate-based probes occurs by in vitro fluorescence measurements and by cell-based or noninvasive animal imaging (right).

cysteine cathepsins (9), and caspases (17), in a substrate-based probe is a fluorogenic probe which multiple proteases have highly similar in which a peptide is coupled to a fluorophore substrate specificity profiles. such as 7-amino-4-carbamoylmethylcoumarin (ACC) or p-nitroaniline (pNA). ACC and pNA are minimally fluorescent when attached to the Substrate-Based Probes probe but become fluorescent when cleaved To measure turnover of natural substrates, from the substrate region (28, 29). Although proteolytic peptides must be isolated and fluorogenic probes are simple in design, probe identified. Substrate-based probes also re- selectivity is limited because the recognition port on substrate hydrolysis, but without sequence occupies only the nonprime side of the need for isolation and characterization the active site. Other types of substrate-based of the peptide products (6). Most synthetic probes do not have this limitation and can have substrate-based probes have two main com- recognition sequences spanning both the non-

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ponents: a recognition sequence that binds prime and prime sides of the substrate-binding in the active site of the protease of interest site (6, 28). For example, substrate-based

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. and a reporter that produces a measurable probes can also be designed to produce a signal signal when the probe is cleaved. The design when a fluorescence resonance energy transfer of the recognition sequence is critical because (FRET) acceptor or fluorescence quencher it is the main component of the probe that (6) is cleaved from the substrate. This optical confers selectivity toward the target protease. signal can be analyzed by plate-reader assay, This sequence is often designed on the basis fluorescence microscopy, and in vivo imaging of natural cleavage sequences or knowledge (Figure 3). Recognition sequences flanked by of substrate specificity obtained from cleavage bioluminescence resonance energy transfer patterns of artificial peptide libraries. The pairs have also been described (30), as have recognition sequence is coupled to a reporter gold nanoparticle–linked probes that change of protease activity. The simplest example of color upon processing by a protease (31).

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Substrate-based probes have also been func- of the protease of interest. An additional lim- tionalized to increase their clinical applicability. itation of substrate-based probes is that after For example, in a recently constructed caspase- cleavage, the substrate diffuses away from the targeted probe, the substrate region is flanked target protease, preventing isolation of the pro- by a Gd3+ chelator and 19F trifluoromethoxy- tease or identification of the precise subcellu- benzylamide. This probe functions as a mag- lar localization of the activity being reported netic resonance imaging (MRI) contrast agent; (6). the intact probe has a low 19F MRI signal due to the paramagnetic effects of Gd3+. Upon sub- strate cleavage, this quenching effect is lost Activity-Based Probes and the probe provides MRI contrast (34). In contrast to substrate-based probes, ABPs co- Substrate-based probes have also been tethered valently attach to active proteases. The covalent to so-called nanoworms that accumulate in dis- bond between a protease and an ABP is formed eased tissues. Protease-mediated cleavage re- between an electrophile on the ABP and the leases a mass reporter that is secreted into the active-site nucleophile of the protease. Alter- urine, which can be collected and detected by natively, a photocrosslinker on the ABP can be mass spectrometry (35). used to covalently label a noncatalytic residue There are several advantages to using within the active site of the protease. Only cat- substrate-based probes to profile protease ac- alytically competent proteases can irreversibly tivity. Various design possibilities exist due to bind to ABPs. As with substrate-based probes, a the simplicity of the scaffold; anything that can recognition sequence confers specificity on the be chemically linked to an amide or a car- target protease. Finally, a reporter tag is ap- boxylic acid can be appended to the substrate. pended to the probe, the nature of which de- Substrate-based probes have also been geneti- pends on the application of interest but is typ- cally encoded through expression of a protease ically either a fluorophore or an affinity handle cleavage sequence flanked by a FRET pair of (6–8). ABP tags can be detected using various fluorescent proteins. This technique has been analytical platforms, including mass spectrome- highly successful for the dynamic study of cas- try, SDS-PAGE, fluorescence microscopy, and pase activation during apoptosis (32). Signal in vivo imaging (Figure 4). amplification is also a potential advantage of The covalent linkage of a probe to the en- substrate-based probes because multiple sub- zyme is the major advantage of activity-based strate molecules can be processed by a single ac- profiling because it enables direct isolation and tive protease. Expanding upon this idea, investi- identification of the target protease. This ability gators have applied macromolecular complexes has facilitated application of ABPs to protease containing many substrate sequences to stud- discovery, prediction of potential off-targets of

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ies of protease function (31, 33). A final advan- drugs, and assignment of the functional roles of tage of substrate-based probes is their utility in closely related proteases (6–8), all of which we

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. high-throughput screening. The frequent use discuss further below. A disadvantage of ABPs is of fluorogenic probes to profile protease sub- that design is somewhat limited by the require- strate specificity and screen for inhibitors has ment for attachment of an electrophile or pho- served as the basis for the design of selective tocrosslinker. Another potential disadvantage inhibitors, substrate-based probes, and ABPs is that, by covalently modifying the protease (6, 28). The major disadvantage of substrate- active site, the target is irreversibly inhibited. based probes is that it remains difficult to as- However, in many cases it is possible to use con- sign substrate cleavage to activation of a partic- centrations of ABP such that only a small per- ular protease. Activation can be inferred only centage of the active enzyme pool is modified from knowledge about the substrate specificity (6, 36).

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a Fluorescently tagged ABP b Affinity-tagged ABP

SH SH

O R R 1 O R O R1 O 3 H 3 H N N N N O N N O H H H H OOR2 OOR2

O O

HO HO

O R R O R R3 1 3 H 1 H N N N N N N S S H H H H OOR OOR2 2 Intensity

m/z

In vivo imaging Blotting/ Mass in-gel fluorescence spectrometry

Figure 4 An example of cysteine protease–targeted activity-based probes (ABPs) with acyloxymethylketone electrophiles. (a) Fluorescently tagged ABPs can be used for in vivo imaging of protease activity and identification of active protease species by in-gel fluorescence. (b) Affinity-tagged (blue star) ABPs can be used to isolate active protease species for identification by immunoblotting or by mass spectrometry. Abbreviation: m/z, mass-to-charge ratio.

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org DESIGNING ACTIVITY-BASED the options for each of the three ABP compo- PROBES FOR PROTEASES nents; we focus on design challenges and how

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. they have been successfully addressed. ABPs have three essential components. The first is a reactive functional group that cova- lently binds to the protease. The second is a linker or recognition sequence that increases se- Reactive Functional Groups lectivity toward target proteases. The third and ABPs contain a reactive functional group that final essential component is a reporter tag by binds covalently to active proteases. For most which the protease-tethered ABP can be visual- cysteine, serine, and threonine protease ABPs, ized or isolated and identified (6–8, 37). Each of the reactive functional group is a directly re- these components is selected to target the pro- active electrophile, whereas for aspartyl pro- tease of interest and to be detected by an opti- tease and metalloprotease ABPs, it is a latent mized analytical platform. This section outlines reactive cross-linker that is activated by light.

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a F of an ABP. Probe labeling is a two-step pro- O F cess in which the probe binds in the protease Peptidyl X Peptidyl active-site cleft and then covalently attaches to O Peptidyl O F O O the active-site nucleophile. The binding step is X: halide O F reversible and is driven by affinity of the probe Halomethylketone Acyloxymethylketone Phenoxymethylketone for the protease, which is measured as the equi-

librium constant, KD. The covalent attachment b step is driven by the reactivity between the nu- OPh F cleophile and the electrophile, which is mea- O Peptidyl Peptidyl Peptidyl P OPh S O P F sured as the rate constant, kcat. Greater elec- O O O trophile reactivity increases the kcat value and Fluorophosphonate Diphenylphosphonate Sulfonyl fluoride lowers the impact of the KD on probe selectiv- ity. In contrast, the rate of modification by less- c d reactive electrophiles is controlled primarily by O the KD, increasing the impact of the substrate- Peptidyl Peptidyl O like region on the specificity of the ABP. How- O O ever, if the rate of reaction is too slow, overall Epoxysuccinate α,β-Unsaturated ester low labeling efficiency may result, hindering ef- O forts to visualize or isolate targets (Figure 6). Peptidyl Peptidyl O S A comparison between two caspase-1-selective OO O O ABPs, FLICA (60) and AWP28 (36), illustrates Epoxyketone Vinyl sulfone this point. Because AWP28 has a lower kcat as well as a lower K for caspase-1 than does Figure 5 D FLICA (36, 68), it displays greater selectivity Representative chemical structures of different classes of electrophiles used in activity-based probes. (a) Activated ketones. (b) Phosphonylating and and lower background labeling (36). This find- sulfonylating agents. (c) Epoxides. (d) Michael acceptors. ing demonstrates that, although various elec- trophiles covalently react with the active-site nucleophile of a protease, electrophiles should Electrophiles for cysteine, serine, and threo- be carefully chosen to maintain ABP specificity. nine protease ABPs have been chosen accord- ing to the reactivity of the target protease. For example, the thiol group in the active site of Challenges in reactive functional group cysteine proteases is more polarizable or “soft” design: aspartyl and metalloproteases. than the hydroxyl group in the active site of Metalloproteases and aspartyl proteases differ serine and threonine proteases (38). There- from cysteine, serine, and threonine proteases

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org fore, electrophiles in cysteine protease ABPs in that they use water as a nucleophile for amide are correspondingly softer than those used for bond hydrolysis. In the few reported examples serine and threonine ABPs (39). Various elec- by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. of ABPs for these classes of proteases, covalent trophile classes are reported to irreversibly in- attachment occurs through a benzophenone hibit cysteine, serine, and threonine proteases or diazirine photocrosslinker (47–50). When (39); a handful of them have been used in ABPs irradiated with UV light, photocrosslinkers (Figure 5)(Table 1) (40–67). produce radical intermediates that react with nearby atoms to form covalent bonds. The Considerations for electrophile selection. higher the binding affinity of the ABP for Electrophiles are often chosen on the basis of the protease is, the more likely that covalent their ease of synthesis, bioavailability, and de- linkage will occur upon UV irradiation. sired reactivity (39). Electrophile reactivity is Importantly, active proteases normally have particularly relevant when tuning the specificity higher ABP binding efficiency than do inactive

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Table 1 Earliest and/or notable applications of electrophiles in activity-based probes (ABPs) for various protease classes Target protease Electrophile type ABP designed for Clan Family Reference(s) Acyloxymethylketone Cysteine Cysteine cathepsins CA C1 40 Legumain CD C13 40 Caspases CD C14 40, 41 Separin CD C50 42 SUMO protease 8 CE C48 43 Alkyne Cysteine Deubiquitinating enzymes CA — 44 Caspases CD C14 44 α,β-Unsaturated ketone Cysteine Cysteine cathepsins CA C1 45 Caspases CD C14 46 Aza-epoxide Cysteine SUMO proteases CE C48 43 Benzophenone Aspartyl, metallo- Metalloproteases — — 47 Presenilin-1 AD A22 48 Diazirine Aspartyl, metallo- Metalloproteases — — 49 Signal peptide peptidase AD A22 50 Diazomethylketone Cysteine Cysteine cathepsins CA — 51 Diphenylphosphonate Serine Serine proteases — — 52 Granzymes A and B PA S1 53 Serine cathepsins PA S1 54 Urokinase-type PA S1 54 plasminogen activator Epoxyketone Threonine Proteasome β-subunits PB T1 55 Epoxysuccinate Cysteine Cysteine cathepsins CA C1 56 Calpain CA C1 57 Caspases CD C14 46 Fluorophosphonate Serine Pan-serine protease — — 58 Halomethylketone Cysteine Cysteine cathepsins CA C1 59 Caspases CD C14 60 SUMO proteases CE C48 61 Phenoxymethylketone Cysteine Papain-fold cysteine CA C1 62 proteases Vinylsulfone Cysteine, threonine Papain-fold cysteine CA C1 63 Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org proteases Deubiquitinating enzymes CA — 64 by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. Proteasome β-subunits PB T1 65 4-Chloroisocoumarin Serine PfSUB1 SB S8 66 Escherichia coli rhomboids ST S54 67

proteases (69). However, photocrosslinking challenge, which has been partially addressed ABPs are still capable of binding to inactive by adding functional groups to the ABP. For proteases because covalent attachment does example, active metalloproteases chelate a not require active-site competency. Therefore, metal ion in their active site. Through the specific binding to active protease forms is a addition of a metal-chelating residue, it is

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site, photocrosslinkers can be located anywhere a along the probe scaffold. It is important to kon + test multiple attachment points because an koff incorrectly placed photocrosslinker can disrupt

kcat interactions with the target protease or indis- criminately react with both inactive and active protease forms (69). A recent study of the as- partyl protease presenilin-1 demonstrated that optimization of photocrosslinking ABPs can be 1 2 used as an opportunity to generate information b high k high k cat cat about the active-site cleft of a protease. In a high KD low KD 1 2 3 4 5 strategy termed photophore walking, investiga- Off-target 1 tors synthesized a panel of ABPs in which a ben- Target protease zophenone photocrosslinker was attached to Off-target 2 three positions of a presenilin-1 inhibitor (48). Labeling Labeling Each position had previously been shown to in- Time Time teract with a different region of the presenilin-1 active-site cleft (71). Protease mutants were ex- 3 4 5

low kcat low kcat optimized pressed that lack activity due to conformational high KD low KD kcat/KD changes in the S2 subsite of the active site. Of the three ABPs, only the ABP containing a pho- tocrosslinker interacting with the S2 subsite se- lectively labeled the wild-type presenilin-1 and Labeling Labeling Labeling not the inactive mutants. The other two ABPs Time Time Time bound to all protease mutants or did not bind to Figure 6 the wild-type protease (48). This observation Kinetic binding and reactivity parameters dictate activity-based probe (ABP) supports the principle that photocrosslinker specificity. (a) ABP–protease interactions have two phases: a reversible binding attachment either can hinder interaction with step determined by the binding affinity (koff /kon,orKD) of the substrate-like the target protease or can enable labeling of specificity region and an irreversible covalent attachment step determined by inactive protease forms. It also highlights the the reactivity of the electrophile (k ). (b) Modulation of ABP K and k to cat D cat need to test different attachment locations. achieve specificity. ABPs with low protease affinity have high KD values. In summary, photocrosslinking ABPs have Highly reactive ABP electrophiles have high kcat values. Numbers 1–5 represent ABPs with various combinations of kcat and KD. For each ABP, there some limitations compared with ABPs for cys- is a rate plot of reaction between the ABP and both target and off-target teine, serine, and threonine proteases because proteases. Lanes 1–5 on the gel (top right) correspond to the sample ABPs; the covalent linkage is not a direct readout of active-

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org bands indicate the labeling intensity of protease species. site competency. However, careful optimiza- tion and validation have generated successful

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. possible to generate ABPs that will selectively ABPs for several aspartyl proteases and metal- label only catalytically active metalloprotease loproteases (47–50). forms (47, 49). This strategy has been validated in cell-culture studies of MMPs and in Xenopus Recognition Sequence Design laevis embryos overexpressing MMP-2 (70). The linker region between the reactive func- An important design consideration for ABPs tional group and the reporter tag is often used of metalloproteases and aspartyl proteases is to confer target specificity to an ABP. the location of the photocrosslinker. Unlike electrophiles in cysteine, serine, and threonine Potential recognition motifs. In some cases, protease ABPs, in which the electrophiles must this linker can be a protein domain or even be positioned directly at the substrate cleavage an entire substrate protein. For example, a

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recently described calpain-targeted ABP con- peptide bicycles could be simultaneously tains a helical calpain recognition domain (57), screened for protease binding and inhibition. and ABPs for deubiquitinating enzymes use the This strategy generated potent and selective full-length ubiquitin as a recognition sequence inhibitors of plasma kallikrein (PK) and factor (72). For most protease-targeted ABPs, the XII (76–78). Notably, the final optimized linker between the reactive group and the tag PK inhibitor had >1,000-fold selectivity for is a peptide sequence that corresponds to the human PK over murine PK. This selectivity sequence of an optimal protease substrate (6). is remarkable, considering the 79% sequence Although optimal recognition sequences identity and 92% sequence similarity between can be determined, a major challenge for the de- the two enzymes (78). sign of the recognition sequence is overlapping Although increasing peptide structural substrate specificity of closely related proteases, diversity will aid in the design of ABPs with which is problematic for imaging applications, exquisite specificity, both of the above ap- the goal of which is to monitor the localization proaches have limitations. Bicyclic peptides and relative activity of a single protease (6–8). are bulky and cell impermeable (76–78), and Several recent advances in substrate screening, although chemical diversity in linear peptide protease inhibitor discovery, and probe design structure is increasing, finding inhibitors with have addressed this problem. absolute selectivity for a native protease re- mains a major challenge (28). To address these Optimizing target specificity. It is often dif- limitations, researchers recently developed a ficult to choose a recognition sequence that protease engineering strategy in which a spe- is specific for a single protease. However, in- cific protease target is engineered to bind with creases in the chemical diversity of substrate absolute specificity to an ABP. This task was screening libraries have facilitated discovery accomplished by introducing an engineered of recognition sequences that discriminate be- cysteine nucleophile near the protease active tween closely related proteases (73). For in- site. A latent electrophile could then be added stance, recently designed fluorogenic substrate to the ABP to make a unique reactive pair and peptide inhibitor libraries are more chemi- (Figure 7). Using this strategy, experimenters cally diverse due to inclusion of both nonnatu- designed ABPs that exclusively target caspase-1, ral and natural amino acids (74). Such a library caspase-8, MMP-12, and MMP-14 (79, 80). was recently used to identify a recognition se- Taken together, these recent advances show quence that has 30-fold selectivity for caspase-3 that it is possible to harness small differences over the closely related caspase-7 protease and between even highly similar proteases to an ABP with 50-fold selectivity for caspase-3 generate ABPs with exquisite specificity. (75). Notably, the pentapeptide recognition se-

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org quence contains three nonnatural amino acids, illustrating the utility of a chemically diverse Reporter Tags

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. screening library. A main advantage of using activity-based profil- Phage display has also been used to generate ing to assess protease activity is the ability to iso- and screen chemically diverse protease in- late and identify targets using various reporter hibitor libraries (76–78). Using phage display, tags. The choice of reporter tag depends on the researchers encoded peptides with constant analytical platform. For instance, ABP imag- cysteine residues and variable spacer regions ing requires fluorescent or radioactive tags, and containing diverse residues. Bicyclic structures ABP-assisted proteomic identification of active reminiscent of peptide macrocyclic drugs were proteases requires affinity handles such as biotin generated by cross-linking the cysteine residues (6–8). However, the choice of tag within the through a tris-(1,3,5-bromomethyl)benzene broad categories of fluorophore and affinity linker. The resulting millions of candidate handle is not trivial, because the tag often

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SH

E E E E

SE E SH E

Covalently labeled Reversibly inhibited protease Reversibly inhibited protease irreversibly inhibited protease

Figure 7 Engineering activity-based probes (ABPs) and proteases to achieve absolute specificity. Engineered ABPs contain reversible and irreversible electrophiles. The reversible electrophile allows positioning within the active site (left, middle). The addition of an irreversible electrophile (E) allows specific covalent linkage to a non-active-site cysteine (SH) on the engineered protease (middle, right).

contributes to the physicochemical properties (84), and a wide variety of fluorescent dyes have of the ABP. Although ABP electrophiles and been synthesized. Among these, BODIPY (85), recognition sequences can be carefully selected fluorescein and rhodamine (86), cyanine dyes to target a protease of interest, they are ineffec- (49), dansyl (87), and NBD (nitrobenz-2-oxa- tive if they do not encounter that protease in 1,3-diazole) (88) have been used in ABPs. The vivo. Incorrect localization can also lead to off- choice of dye usually depends on the desired target labeling. For example, improper biodis- chemical properties of the resulting probe. For tribution has been a major challenge when de- example, fluorescein and rhodamine have poor signing ABPs for cytoplasmic proteases because photostability and should not be used for ex- many ABPs are hydrophobic, charged, or bulky periments that require extended imaging time and therefore are not cell permeable. Cell- (86, 89), but they are inexpensive and thus an impermeable ABPs are often endocytosed and economical choice for other applications. label predominantly endosomal and lysosomal Although many dyes may lack optimal proteases (83). ABPs can also suffer from lack chemical or biological properties, their charac- of solubility, which limits their biodistribution. teristics can be improved by derivatization. For

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org Fortunately, the increasing diversity of reporter example, esterification of fluorescein quenches tags has correspondingly allowed for optimiza- its fluorescence until it enters the cell and is

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. tion of ABP efficiency. Considerations for tag liberated by cytosolic esterases (89). If a dye selection are discussed in detail below. has high in vivo background, it can be paired with a quencher that absorbs its fluorescence Considerations for selection of an imag- until the ABP is bound to the target protease. ing tag. Although ABPs are known as small- This method has been used successfully for in molecule probes, they often are not classically vivo imaging of protease activity (Figure 8). defined as small molecules but rather can have Quenchers can also be optimized; for example, masses approaching 10 kDa (82, 83). Much of sulfonation of the quencher Qsy21 increases this mass is often the reporter tag, especially its solubility and biodistribution. Applying this when the tag is a dye. Small-molecule dyes modification to a pan-cathepsin ABP resulted have long been employed in biological research in significant improvements in tumor contrast

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in an orthotopic mouse model of breast cancer (90). a b

Considerations for selection of an affin- ity handle. Many factors should be taken into –S –S account when choosing an appropriate ABP affinity handle. Typical choices include biotin, hemagglutinin (HA), azides, and alkynes (6–8, 37, 72). Biotin- and HA-tagged ABPs can be S used to isolate labeled targets by affinity pu- S rification. Azides and alkynes react with one another in a copper-catalyzed click reaction to form a triazole (91). These two functional c groups can be used as latent reporter tags on ABPs that can be converted into affinity probes by use of the corresponding azide or alkyne tags. Tumor Direct biotin or HA tagging is advantageous because it allows direct isolation of a labeled tar- get. However, these tags generally reduce cell permeability. A “clickable” handle often does not hinder the permeability of an ABP, but iso- lation depends on efficiency of the click reaction and subsequent isolation steps (92). Both meth- ods of affinity purification have been success- fully used to identify drug targets and discover new enzymes (72). In general, HA or biotin Figure 8 tags are useful if protein isolation is performed Quenched activity-based probes (ABPs) have improved contrast relative to in cell lysates and if these relatively significant unquenched ABPs in noninvasive imaging experiments. (a) Unquenched probes are constitutively fluorescent. (b) Quenched probes are not fluorescent until modifications to the ABP do not prohibit bind- cleavage of the fluorescence quencher (red octagon) occurs upon binding to ing to the target protease. In other cases, a click- protease. (c) Quenched ABPs have lower background fluorescence than able handle may be more effective (37, 72). unquenched ABPs, as demonstrated by this comparison of in vivo ABP labeling of tumor-associated cathepsins by an unquenched ABP (left) and a quenched ABP (right). Modified with permission from Reference 83. APPLICATIONS OF ACTIVITY-BASED PROFILING all capable of inducing protease activation by

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org TO PROTEASES altering the conformation of or access to the Advances in probe design have facilitated ap- active site (2). Of the methods of profiling pro-

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. plications of ABPs to the study of fundamental tease activity, substrate-based probes are the protease enzymology, to the discovery and de- most commonly used to determine the require- velopment of drugs, and to the diagnosis of dis- ments for protease activation. For example, ease (6–8). In the following sections, we discuss pNA-linked peptides were used to establish the these ABP applications by highlighting case effect of amino acid point mutations on binding studies that best illustrate each application. of the serine protease factor VIIa to its allosteric activator, tissue factor (93, 94). ABPs have also been successfully used to Protease Enzymology study mechanisms of protease activation. The Proteolytic processing, cofactor binding, post- main advantage of using ABPs is that the pro- translational modification, and pH change are tease forms that are active can be identified.

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In contrast, measurements of substrate hydro- losterically activates the full-length caspase-7 lysis only designate whether a protease is ac- prior to completion of proteolytic processing. tive under the conditions of interest; they do This finding was more recently supported by not provide insight into which protease forms structural studies of caspase-7 mutants bound are capable of hydrolyzing substrates. ABPs can to covalent active-site inhibitors (98). Impor- identify all active protease forms, an ability that tantly, monitoring caspase-7 activation by im- has been particularly advantageous for the iden- munoblotting or fluorogenic substrate assay tification of transiently active protease forms would not have provided this insight, support- (96, 97). Another advantage of using ABPs to ing the concept that ABPs are useful for trap- study protease enzymology is that covalent at- ping and identifying transiently present active tachment of ABP to protease depends on the protease forms. catalytic competency of the active site and not Profiling allosteric activation of the its substrate-binding ability. This advantage is Clostridium difficile TcdB cysteine protease useful for identifying protease mutations that domain. Clostridium difficile is a gram-positive restrict access to the active-site cleft without al- bacterium that is a major cause of hospital- tering its conformation (95, 96). The following acquired diarrhea. The glucosylating toxins case studies expand on these issues and illustrate TcdA and TcdB are the primary virulence how ABPs can be used to determine details of factors of this pathogen (99). TcdB contains protease activation that are difficult to identify a cysteine protease domain (CPD) that is using other techniques. activated by allosteric binding of the host cell cofactor inositol hexakisphosphate (InsP ) Intermediates of caspase-7 activation. 6 (100). Because the primary function of the Caspase-7 is a cysteine protease that is activated CPD is autoprocessing of the full-length toxin, during the programmed cell death process, ap- it does not efficiently bind substrate in trans optosis (12, 17). Its activation was originally and therefore does not process short peptide thought to occur by limited proteolysis coordi- fluorogenic substrates (101). Therefore, an nated by related family members caspase-3 and ABP specific for TcdB was required to detect caspase-9. Furthermore, only the mature, pro- activation of the CPD in response to InsP . cessed form of caspase-7 was thought to have 6 Surprisingly, the recombinant TcdB CPD proteolytic activity. Caspase-7 activation had could be labeled by ABP in the absence of InsP . previously been measured by immunoblotting, 6 This finding supports a dynamic structure of which allowed direct observation of the con- the CPD that samples active conformations version of the 36-kDa precursor enzyme to the even without the activating cofactor bound. 20-kDa mature form, and by fluorogenic sub- The authors of this study proposed that the strate assay. Alternatively, an ABP was success- ABP served as a substrate mimic that stabilized Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org fully used to identify incompletely processed the active conformation of the protease, which forms of caspase-7 that have transient activ- was supported by limited proteolysis experi- by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. ity. In this study, the apoptotic caspase cas- ments (97). This intermediate could not have cade was induced in a cell-free system by cy- been measured by fluorogenic substrate assays tochrome c and dATP. Activated caspases were due to lack of sensitivity and the transient labeled by a pan-caspase ABP. Upon stimu- nature of the interaction between a substrate lation, the mature form of caspase-7 and the and the enzyme, yet it could be readily isolated full-length, uncleaved “zymogen” form were la- and identified using a covalently bound ABP beled by the ABP (96). A further analysis in- (Figure 9). dicated that the full-length species is part of a heterodimeric complex composed of mature, Activity-based profiling of the Escherichia processed caspase-7 and full-length, zymogen coli rhomboid protease GlpG. The rhom- caspase-7 in which the mature caspase-7 al- boid proteases are a family of intermembrane

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serine proteases with proposed roles in cell sig- a naling, cell death, and host–pathogen interac- tions. These proteases cannot be easily purified for functional studies, which makes biochemi- cal characterization challenging. The structure Cofactor-bound of the Escherichia coli rhomboid protease GlpG active protease was recently determined (102). However, the structures did not include the cytoplasmic do- main of the protease. To circumvent purifica- tion difficulties, the activities of mutants of the Inactive Active cytoplasmic domain were determined in crude Conformation sampling membrane extracts. Substrate-based probe screening revealed an α-helix in the cytoplas- mic domain that is important for GlpG activity. b Specific mutations were made in this α-helix and screened for activity by use of a substrate- based probe and an ABP. Interestingly, a mutation was identified that showed complete loss of fluorogenic substrate processing but was labeled by the ABP. The ABP did not have Full-length Full-length Mature a peptidic specificity region but instead had a inactive active active small alkyl linker, which led to the hypothesis Figure 9 that the protease mutant could no longer bind Activity-based probes can be used to capture latent active forms of proteases. to native substrates but still had a catalytically (a) A protease that requires cofactor binding ( green pentagon) for full activity competent active-site nucleophile (95). This can also transiently assume an active conformation when unbound. (b)Many finding demonstrates that ABPs can distinguish proteases must undergo proteolytic processing to become active and pass mutations that alter substrate-binding ability through a series of intermediate truncation steps. Here, processing is promoted from those that alter catalytic activity. It also by allostery. Allosteric binding supports an active conformation of the incompletely processed protease (middle). illustrates the value of using complementary methods to measure protease activity. suitability for high-throughput screening. As an alternative, investigators developed a method termed fluopol-ABPP that uses Drug Discovery and Development fluorescent ABPs to enable high-throughput Dissecting the activation mechanism of a screens (103). This technique uses fluorescence protease can provide insight into potential polarization to detect binding of a fluorescent

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org mechanisms by which it can be artificially ABP to an active protease. When an ABP modulated. The ability to manipulate protease is bound to the target, the fluorophore tag

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. activity is useful for mapping biological func- “tumbles” more slowly in solution, resulting in tion. It is also clinically useful because proteases enhanced fluorescence polarization. However, are aberrantly active in many disease patholo- when binding of an inhibitor blocks labeling, gies, and inhibition is often a viable method of the unbound ABP rotates quickly in solution treatment (4). and fluorescence polarization is reduced. By Protease inhibitors and activators are often measuring the change in the fluorescence identified by screening compounds against polarization value, one can assess the ability of enzymes purified from a native source or en- a compound to bind and inhibit a target pro- zymes isolated from recombinant expression in tease. The benefit of this method is that it does bacteria. Fluorogenic substrates are commonly not require a substrate, is highly amenable to used to assess target inhibition due to their high-throughput screening, and is less affected

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by artifacts that result from intrinsic fluores- screen was conducted in the native environ- cence and poor solubility of the compounds ment of the protease, and covalent attachment being screened. Fluopol-ABPP has been used of the drugs enabled verification of target to identify selective and potent inhibitors of engagement and analysis of off-target binding. the serine protease PREPL, as well as several other serine hydrolases (SHs) (104, 105). Trypanocide development. The trypano- Although useful, such screens require that somes Trypanosoma cruzi and Trypanosoma bru- the protease of interest be isolated in pure form. cei are human parasites that cause Chagas dis- In addition, these types of in vitro screens do not ease and African sleeping sickness, respectively. provide any information about the in vivo po- Existing treatments for these diseases are toxic tency or selectivity of the identified lead com- and often ineffective, and drug resistance is in- pounds. However, such characteristics can be creasing (108). The cysteine proteases cruzain assessed by competitive ABPP, in which a com- and rhodesain are essential for the parasites plex proteome or intact cell is first treated with (109), making them promising drug targets. a drug candidate and then labeled by an ABP. In a recent study, fluorogenic substrates were This approach is advantageous because screens used to identify a novel class of inhibitors can be performed in a physiologically relevant with azanitrile electrophiles. Of the lead com- setting. Potency is measured by assessing loss pounds, two were converted into ABPs by tag- of ABP labeling of the target protease over a ging the carboxybenzyl cap of the inhibitors titration of the lead compound (7, 8). An esti- with an alkyne. Click chemistry–enabled con- mate of selectivity can also be determined if the jugation of azido-rhodamine to the alkyne al- ABP can label related enzymes (8). The follow- lowed visualization of probe labeling by in-gel ing examples demonstrate the applicability of fluorescence and characterization of compound ABPs to drug discovery and development. uptake and subcellular distribution by fluores- cence microscopy. Reaction with azido-biotin Discovering inhibitors of the Clostridium facilitated immunoprecipitation and identifica- difficile protease Cwp84. In addition to tion of ABP-bound proteases. In both cases, killing host cells through the CPD-containing the ABPs confirmed binding to off-target pro- toxins TcdA and TcdB, C. difficile infection is teins such as ketoacyl-CoA thiolase, BILBO1, promoted by another protease, Cwp84. Cwp84 and succinyl-CoA synthetase α, all of which are is a recently identified papain family cysteine essential to parasites. Therefore, the authors protease that is responsible for generating the speculated that the off-target binding increased polypeptide surface layer that coats C. difficile. the lethality of their compounds. Although ge- This surface layer protects C. difficile from netic validation is required to prove this claim host defenses and aids in the invasion process (110), this case study shows the complemen-

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org (106). Cwp84 is a potential drug target because tarity of substrate-based probes and ABPs and its inhibition simultaneously facilitates host demonstrates the value of ABPs for dissecting

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. immune system activation and blocks bacterial off-target effects of drugs. invasion of host cells. Because Cwp84 is a papain family cysteine protease, a panel of Pharmacodynamics of cathepsin K inhibi- potential inhibitors was synthesized on the tors. Cathepsin K is a cysteine protease that basis of the structure of the well-known papain is involved in bone resorption, the process by inhibitor E-64 (107). This library was screened which osteoclasts degrade bone. This process by competitive ABPP in C. difficile cell lysates. is important for increasing calcium levels in the The top candidate, an E-64 analog with an blood, but excessive bone resorption can make α,β-unsaturated ester electrophile, was the bones brittle (111). For this reason, cathepsin K most potent and displayed little off-target has become an important drug target for treat- binding in bacterial cell lysates. Notably, this ment of osteoporosis. A recently developed

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class of cathepsin K inhibitors containing a activity-based profiling coupled with mass spec- piperazine ring failed to progress to the clinic trometry has proven to be a good source of due to toxicity issues. Notably, analogous novel disease biomarkers (116). inhibitors lacking the piperazine functional Once a biomarker is identified, methods group were nontoxic (112). An ABP was used must be developed for reliable clinical detec- to analyze the specificity and distribution of tion. Such methods must be able not only to these two classes of compounds, with the detect protease activity, but also to distinguish aim of explaining the observed differences in healthy tissue from diseased tissue. ABPs toxicity. To analyze the effects of both groups have been successfully used to detect aberrant of inhibitors on cysteine cathepsin activity, protease activity in various patient samples. female rats were treated with the compounds These include brain tumor tissue, in which and then treated with a pan-cysteine cathepsin fluorescent ABPs were topically applied and ABP. Tissues were excised and cathepsin in- imaged (117), and blood, in which peripheral- hibition assessed by in-gel fluorescence. In all blood mononuclear cells were labeled with the tissues analyzed, the cathepsin K inhibitors an ABP, separated by cell type, and analyzed containing a piperazine ring inhibited the by in-gel fluorescence (118). ABPs have also off-target cathepsins B, S, and L, whereas the been used to noninvasively image areas of other inhibitors did not. The authors of this inflammation and cancer in various animal study hypothesized that off-target inhibition models (81, 83). These applications and the was due to protonation of the basic nitrogen use of ABP to identify novel biomarkers are in the piperazine ring and accumulation in the discussed in the following case studies. lysosome, which increases binding to lysosomal cathepsins B, S, and L. This study was valuable Identification and validation of myelobla- because it demonstrated that nonbasic com- stin as a non-small-cell lung cancer bio- pounds have increased selectivity for cathepsin marker. The prognosis of non-small-cell lung K due to better distribution properties, which cancer (NSCLC) is based on the stage of the should improve the safety and efficacy of future disease at diagnosis, which often is not predic- cathepsin K–targeted compounds (113). Fur- tive of the disease course. Therefore, clinically thermore, it illustrates an application of ABPs in relevant biomarkers of NSCLC are needed. the drug-development process and emphasizes An ideal biomarker would display significant the importance of being able to monitor drug changes in activity that correlates with disease selectivity and distribution dynamics in vivo. onset and progression. Because differential SH activity has previously been associated with multiple types of cancer (119), SHs are candi- Profiling Proteases in Disease date biomarkers for NSCLC. To profile a wide

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org In addition to being viable targets for treatment and relatively unbiased range of potential SH of disease, proteases are also potential biomark- biomarkers, biopsies from both healthy patients

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. ers that can be used for diagnostic purposes. For and patients with lung adenocarcinoma, the example, proteases are ideal biomarkers for in- most common type of NSCLC, were labeled flammatory diseases because they are secreted with a pan-SH ABP. Labeled proteases were from activated immune cells (114). They are affinity-purified with streptavidin and identified also useful biomarkers for cancer because in- using mass spectrometry. Using this method, creased protease activity is associated with many investigators identified 33 SHs that had sig- of the hallmark processes of cancer, includ- nificantly elevated activity in tumor biopsies. ing angiogenesis, tissue remodeling, and cell Notably, 9 of the 33 SHs were serine pro- death (115). Specific protease biomarkers are teases (120). In a follow-up study, the authors often identified from gene-expression profiling pursued myeloblastin, a serine protease, as a or shotgun proteomic data (8). More recently, potential biomarker for KRAS-driven NSCLC

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(121). Upregulation of myeloblastin activity in ABPs for imaging protease activity, namely that tumors from NSCLC patients with a G12C the specific proteases labeled by an ABP can be mutation in KRAS was established by ABP- identified. enabled affinity purification. Although these findings need to be repeated in more patients Activity-based probe–based methods to in order to effectively validate myeloblastin determine kallikrein 6 activity in patient as a biomarker, they demonstrate the appli- samples. Kallikreins (KLKs) are serine pro- cability of ABPP for biomarker discovery and teases that have trypsin- or chymotrypsin-like validation. On the basis of the low sample size activity. Within this family, KLK3 is a widely of the study, it is also impressive that coherent used biomarker for ovarian cancer. KLK6 is trends in protease activity could be identified, also a potential biomarker because it activates which illustrates the value of profiling protease proteinase-activated receptors, leading to activity within a native cellular environment. pathologies such as inflammation, carcinogen- esis, and tumor metastasis. KLKs have elevated Noninvasive imaging of cathepsin S. Cys- expression in tumors but differential expression teine cathepsins are lysosomal proteases that in metastatic and primary tumors, suggesting change subcellular distribution and activity in that they are markers of tumor progression conditions such as inflammation and cancer. (124). However, diagnostic assays are required Cysteine cathepsins are useful targets for imag- to verify that KLK6 can function as a cancer ing sites of cancer and inflammation because biomarker and to identify KLK6 activity in their expression is elevated in the macrophages relevant tissues or fluids. To this end, an ABP- that infiltrate many types of diseased tissues. mediated assay was developed to detect KLK6 Several reported classes of ABPs label cysteine in biological fluids from cancer patients. In this cathepsins B, L, and S (40, 83, 122). How- assay, termed ABRA-ELISA (ABP ratiometric ever, although the expression of cathepsins B, enzyme-linked immunosorbent assay), relevant L, and S is elevated in macrophages, cathep- biological fluids were collected and incubated sins B and L are also active in the surround- with a biotinylated ABP. The ELISA for KLK6 ing tissues. In contrast, cathepsin S is expressed reported the total enzyme levels, and the ELISA only in antigen-presenting cells (118), suggest- for biotin reported active enzyme levels. The ing that an ABP that exclusively labels cathep- results of both assays were combined to suc- sin S might have improved contrast between cessfully quantify the percentage of total KLK6 diseased tissue and healthy tissue. Researchers that is active in cerebrospinal fluid, ascites designed an ABP containing a cathepsin S– fluid, and cancer cell supernatants. In most selective nonpeptidic specificity region (122, samples, only ∼5% of the expressed KLK6 was 123). Importantly, this ABP showed improved active (125). The biotin ELISA had sufficient

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org tumor contrast compared with pan-cathepsin sensitivity to identify this low level of activity, ABPs. Cathepsin S–selective labeling was veri- but the assay could not have been performed

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. fied by in-gel fluorescence (123), again demon- without covalent linkage of the ABP to the strating one of the major advantages of using protease.

SUMMARY POINTS 1. Levels of protease activity more accurately define function than do protease messenger RNA or protein quantities. 2. Protease activity can be determined by measuring hydrolysis of natural substrates and substrate-based probes or covalent attachment of ABPs.

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3. The various methods of profiling protease activity have distinct advantages and are best used complementarily. 4. The three major elements of an ABP—the reactive functional group, specificity region, and reporter tag—are chosen on the basis of the target protease and the desired analytical platform. 5. ABPs can be used to identify intermediate species in protease activation and to distinguish factors altering catalytic activity from those obstructing access to the active site. 6. A major advantage of applying ABPs to drug discovery and development is that the in vivo potency and selectivity of drug candidates can be simultaneously screened. 7. Protease ABPs have also been successfully applied to the discovery, validation, and imag- ing of disease biomarkers.

FUTURE ISSUES 1. Systems-level substrate profiling should be used to determine fine differences in substrate specificity between closely related enzymes. 2. ABPs usually react with multiple proteases, which is problematic for imaging studies. Chemically diverse inhibitor libraries and phage methods may increase the selectivity of future ABPs. 3. Improvements in synthesis and derivatization of dyes and affinity handles should be applied to further improve ABP biodistribution, target engagement, and selectivity. 4. Incorporation of novel electrophiles into ABPs will enable targeting of broader classes of proteases and allow for better control of specificity of existing probes. 5. The clinical applicability of ABPs can be improved by addition of reporter tags facilitat- ing detection by positron emission tomography, MRI, or other commonly used clinical methods.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org might be perceived as affecting the objectivity of this review. by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. ACKNOWLEDGMENTS The authors thank Matthew Child, Nimali Withana, and Ian Foe for helpful discussions about this manuscript. This material is based on work supported by the National Science Foundation under grant DGE-114747 (to L.E.S.) and by the National Institutes of Health under grant R01- EB005011 (to M.B.).

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Annual Review of Biochemistry Contents Volume 83, 2014

Journeys in Science: Glycobiology and Other Paths Raymond A. Dwek ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1 Lipids and Extracellular Materials William Dowhan pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp45 Topological Regulation of Lipid Balance in Cells Guillaume Drin ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp51 Lipidomics: Analysis of the Lipid Composition of Cells and Subcellular Organelles by Electrospray Ionization Mass Spectrometry Britta Br¨ugger ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp79 Biosynthesis and Export of Bacterial Lipopolysaccharides Chris Whitfield and M. Stephen Trent pppppppppppppppppppppppppppppppppppppppppppppppppppppp99 Demystifying Heparan Sulfate–Protein Interactions Ding Xu and Jeffrey D. Esko ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp129 Dynamics and Timekeeping in Biological Systems Christopher M. Dobson pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp159 Metabolic and Nontranscriptional Circadian Clocks: Eukaryotes Akhilesh B. Reddy and Guillaume Rey ppppppppppppppppppppppppppppppppppppppppppppppppppppp165 Interactive Features of Proteins Composing Eukaryotic Circadian Clocks Brian R. Crane and Michael W. Young ppppppppppppppppppppppppppppppppppppppppppppppppppp191

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org Metabolic Compensation and Circadian Resilience in Prokaryotic Cyanobacteria

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. Carl Hirschie Johnson and Martin Egli pppppppppppppppppppppppppppppppppppppppppppppppppppp221 Activity-Based Profiling of Proteases Laura E. Sanman and Matthew Bogyo pppppppppppppppppppppppppppppppppppppppppppppppppppp249 Asymmetry of Single Cells and Where That Leads Mark S. Bretscher ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp275 Bringing Dynamic Molecular Machines into Focus by Methyl-TROSY NMR Rina Rosenzweig and Lewis E. Kay pppppppppppppppppppppppppppppppppppppppppppppppppppppppp291

v BI83-Front-Matter ARI 30 April 2014 10:15

Chlorophyll Modifications and Their Spectral Extension in Oxygenic Photosynthesis Min Chen pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp317 Enzyme Inhibitor Discovery by Activity-Based Protein Profiling Micah J. Niphakis and Benjamin F. Cravatt pppppppppppppppppppppppppppppppppppppppppppppp341 Expanding and Reprogramming the Genetic Code of Cells and Animals Jason W. Chin ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp379 Genome Engineering with Targetable Nucleases Dana Carroll pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp409 Hierarchy of RNA Functional Dynamics Anthony M. Mustoe, Charles L. Brooks, and Hashim M. Al-Hashimi pppppppppppppppppp441 High-Resolution Structure of the Eukaryotic 80S Ribosome Gulnara Yusupova and Marat Yusupov pppppppppppppppppppppppppppppppppppppppppppppppppppp467 Histone Chaperones: Assisting Histone Traffic and Nucleosome Dynamics Zachary A. Gurard-Levin, Jean-Pierre Quivy, and Genevi`eve Almouzni pppppppppppppp487 Human RecQ Helicases in DNA Repair, Recombination, and Replication Deborah L. Croteau, Venkateswarlu Popuri, Patricia L. Opresko, and Vilhelm A. Bohr ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp519 Intrinsically Disordered Proteins and Intrinsically Disordered Protein Regions Christopher J. Oldfield and A. Keith Dunker ppppppppppppppppppppppppppppppppppppppppppppp553 Mechanism and Function of Oxidative Reversal of DNA and RNA Methylation Li Shen, Chun-Xiao Song, Chuan He, and Yi Zhang pppppppppppppppppppppppppppppppppppp585 Progress Toward Synthetic Cells Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org J. Craig Blain and Jack W. Szostak ppppppppppppppppppppppppppppppppppppppppppppppppppppppp615

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. PTEN Carolyn A. Worby and Jack E. Dixon ppppppppppppppppppppppppppppppppppppppppppppppppppppp641 Regulating the Chromatin Landscape: Structural and Mechanistic Perspectives Blaine Bartholomew ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp671 RNA Helicase Proteins as Chaperones and Remodelers Inga Jarmoskaite and Rick Russell pppppppppppppppppppppppppppppppppppppppppppppppppppppppppp697

vi Contents BI83-Front-Matter ARI 30 April 2014 10:15

Selection-Based Discovery of Druglike Macrocyclic Peptides Toby Passioura, Takayuki Katoh, Yuki Goto, and Hiroaki Suga ppppppppppppppppppppppppp727 Small Proteins Can No Longer Be Ignored Gisela Storz, Yuri I. Wolf, and Kumaran S. Ramamurthi ppppppppppppppppppppppppppppppp753 The Scanning Mechanism of Eukaryotic Translation Initiation Alan G. Hinnebusch ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp779 Understanding Nucleic Acid–Ion Interactions Jan Lipfert, Sebastian Doniach, Rhiju Das, and Daniel Herschlag pppppppppppppppppppppp813

Indexes Cumulative Index of Contributing Authors, Volumes 79–83 ppppppppppppppppppppppppppp843 Cumulative Index of Article Titles, Volumes 79–83 ppppppppppppppppppppppppppppppppppppp847

Errata An online log of corrections to Annual Review of Biochemistry articles may be found at http://www.annualreviews.org/errata/biochem Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only.

Contents vii Annual Reviews It’s about time. Your time. It’s time well spent.

New From Annual Reviews: Annual Review of Statistics and Its Application Volume 1 • Online January 2014 • http://statistics.annualreviews.org Editor: Stephen E. Fienberg, Carnegie Mellon University Associate Editors: Nancy Reid, University of Toronto Stephen M. Stigler, University of Chicago The Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that allow for their implementation. It will include developments in the field of statistics, including theoretical statistical underpinnings of new methodology, as well as developments in specific application domains such as biostatistics and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical sciences. Complimentary online access to the first volume will be available until January 2015.

table of contents: • What Is Statistics? Stephen E. Fienberg • High-Dimensional Statistics with a View Toward Applications • A Systematic Statistical Approach to Evaluating Evidence in Biology, Peter Bühlmann, Markus Kalisch, Lukas Meier from Observational Studies, David Madigan, Paul E. Stang, • Next-Generation Statistical Genetics: Modeling, Penalization, Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage, and Optimization in High-Dimensional Data, Kenneth Lange, Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema, Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobel Patrick B. Ryan • Breaking Bad: Two Decades of Life-Course Data Analysis • The Role of Statistics in the Discovery of a Higgs Boson, in Criminology, Developmental Psychology, and Beyond, David A. van Dyk Elena A. Erosheva, Ross L. Matsueda, Donatello Telesca • Brain Imaging Analysis, F. DuBois Bowman • Event History Analysis, Niels Keiding • Statistics and Climate, Peter Guttorp • Statistical Evaluation of Forensic DNA Profile Evidence, • Climate Simulators and Climate Projections, Christopher D. Steele, David J. Balding Jonathan Rougier, Michael Goldstein • Using League Table Rankings in Public Policy Formation: • Probabilistic Forecasting, Tilmann Gneiting, Statistical Issues, Harvey Goldstein Matthias Katzfuss • Statistical Ecology, Ruth King

Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org • Bayesian Computational Tools, Christian P. Robert • Estimating the Number of Species in Microbial Diversity • Bayesian Computation Via Markov Chain Monte Carlo, Studies, John Bunge, Amy Willis, Fiona Walsh

by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. Radu V. Craiu, Jeffrey S. Rosenthal • Dynamic Treatment Regimes, Bibhas Chakraborty, • Build, Compute, Critique, Repeat: Data Analysis with Latent Susan A. Murphy Variable Models, David M. Blei • Statistics and Related Topics in Single-Molecule Biophysics, • Structured Regularizers for High-Dimensional Problems: Hong Qian, S.C. Kou Statistical and Computational Issues, Martin J. Wainwright • Statistics and Quantitative Risk Management for Banking and Insurance, Paul Embrechts, Marius Hofert

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